Last year's annual report summarized involvement of ppGpp in diauxic growth lags during changes of carbon sources. These shifts trigger complex adjustments of gene expression and post-translational protein acetylation during exhaustion of glucose to growth on other sugars. This work has now been published Fernndez-Coll L, Cashel M. (2018) Front Microbiol. 9:1802. PMID:30123210. In summary, complex regulation of (p)ppGpp is documented to arise from carbon starvation effects on hydrolase, amino acid starvation effects on synthesis and reabsorption of secreted acetate to reverse the normal pathway for making acetyl phosphate (Ac-P) from acetyl-CoA. As ppGpp increases, so does the source of Ac-P the source for posttranslational acetylation and phoshorylation. This study required development of high throughput isotope assays of changing (p)ppGpp levels that led to a video publication of the method. Fernndez-Coll L, Cashel M. 2019. J Vis Exp. 148. PMID:31233015. This year we extended last year's biochemical comparison of (p)ppGpp and (p)ppApp interactions with E. coli RNA polymerase and regulatory activities using a standard ppGpp inhibited E. coli promoter Bruhn-Olszewska B, Molodtsov V, Sobala M, Dylewski M, Murakami KS, Cashel M, Potrykus K. (2018) Biochim Biophys Acta Gene Regul. Mech. 1861(8):731-742. PMID:30012465. The RNAP binding site of (p)ppApp is near the catalytic center near to, but distinct from, the two known (p)ppGpp binding sites Molodtsov V, Sineva E, Zhang L, Huang X, Cashel M, Ades SE, Murakami KS. (2018) Mol Cell. 69:828-839. PMID: 29478808. Our initial focus was on the catalytically bifunctional ppGpp synthetase/hydrolase RSH enzyme. It was proven that this pure protein synthesizes (p)ppApp as well as (p)ppGpp. This RSH enzyme was found in an unusual Methylobacterium species (Mext) discovered by Dr. Potrykus to also harbor an unusual small hydrolase that cleaves both (p)ppGpp and (p)ppApp. Induced ectopic expression of the RSHMext in E. coli led to detection of appreciable (p)ppApp, which required development of a new TLC detection method. Surprisingly, E. coli controls that lacked the ectopic RSHMext protein also revealed traces of (p)ppApp with the new assay. This is the first observation of (p)ppApp in E. coli. Evidence was also obtained suggesting that induced RSHMext-mediated (p)ppApp accumulation has regulatory consequences in E. coli. Sobala M, Bruhn-Olszewska B, Cashel M, Potrykus K. (2019) Front Microbiol. 10:859. PMID:31068922. This now leads to a search in E. coli for physiological conditions that provoke (p)ppApp accumulation and asking if cellular (p)ppApp might function to antagonize (p)ppGpp regulation as suggested in vitro. Interestingly, not all RSH enzymes synthesize or hydrolyze (p)ppApp; we have past experience with a Streptococcal RSHSeqi whose synthesis and hydrolase activities are limited to G- and not A-analogs. A future goal is to exploit the Mext RSH protein and its catalytic fragments in an attempt to achieve incremental accumulation of only ppApp or pppApp in order to assign new functions for each and explore their possible interactions. It is noteworthy that the RNAP binding sites for (p)ppApp are found in both Gram-positive and -negative bacteria whereas direct (p)ppGpp regulatory interactions with RNAP are absent in Gram positives. This suggests another level of diversity exists for roughly half of the bacterial kingdom. In past annual reports we have described attempts to unravel the how (p)ppGpp determines rates of balanced exponential bacterial growth. As classically defined 60 years ago, growth rates are said to be balanced when they are determined by nutrient composition rather than by limited abundance. This caveat is important because optimal efficiency of nutrient use requires fine tuning of gene expression in contrast to starvation where adjustments avoid death. Therefore starvation for sources of carbon, nitrogen or phosphate, whether due to abrupt nutrient removal, steady state chemostat or turbidostat limitations, results in physiological consequences are very different from fine tuning. We recently published a commentary on differences between starvation and balanced growth Potrykus K, Cashel M. (2018) Nature Microbiol. 3:862-863, PMID:30046172. Long ago it was hypothesized that the number of cellular ribosomes, all operating to produce proteins at high efficiency determined balanced growth rates. This has since been largely validated with ppGpp as the key. Our interest in ppGpp as a regulator of balanced growth was piqued when isolated (p)ppGpp hydrolase mutants were found to grow progressively slower with 2X, 4X, 8X and 12X incremental increases of (p)ppGpp basal levels. Importantly, the correlation between slower balanced exponential growth rates and increased basal levels occurred despite the lack of physiological stress. This indicated (p)ppGpp levels are themselves sufficient to regulate growth rates, but how? Several labs, including our own, worked on the details of ppGpp inhibition of transcription from ribosomal RNA promoters as an obvious way to regulate ribosomal content. Our next step carried out by Dr. Potrykus, then a postdoc, was to study deletion mutants that completely lack (p)ppGpp, termed ppGpp0. These mutant cells displayed consistently high levels of cellular rRNA and ribosome content otherwise typical of balanced fast growth even during slow growth on inefficient media. The absence of regulation when ppGpp is missing indicated that ppGpp is necessary for controlling growth rates. Finally RNAP mutant suppressors of ppGpp0 strains that phenotypically mimic the presence of ppGpp even in its absence were found to also slow growth and appropriately lower the otherwise high levels of rRNA. In summary, this suggests ppGpp is necessary and sufficient to control growth exerted by as yet unknown effects on transcription. Manipulations that resulted in accumulation of either ppGpp or pppGpp revealed ppGpp to be a more potent regulator of growth rate of the two, a recurrent theme for other regulatory motifs. Current parallel experiments now reveal that ppGpp is necessary and sufficient to regulate initiation of chromosomal DNA replication also as a function of different balanced growth rates. Bacteria can grow very quickly but it takes longer for bacteria to duplicate their DNA than to divide. They resolve this disparity by multiple bidirectional initiations from a single origin (ori) region that occur before DNA replication is completed at a single terminator (ter) sequence. Measuring ori/ter ratios by PCR provides accurate estimates of even small differences of initiation frequencies. It is known that ppGpp also mildly inhibits DNA elongation through primase. Slowing of elongation at specific sites by ppGpp have been ruled out by genomic sequencing of fast and slow growing Wt and ppGpp0 strains. Flow cytometric studies by our collaborators in the Szaweleska lab show that immature chromosomes also fail to support an explanation at the level of elongational pausing. For wild type (WT) cells, rates of DNA initiation measured as ori/ter ratios drop about 3-fold comparing fast to slow growth, while the ori/ter ratios of ppGpp0 cells remain constantly high even when their rate of balanced growth is slow. SpoT hydrolysis mutants with high ppGpp basal levels also lower ori/ter ratios to match those of slow growing WT cells. Finally RNAP suppressor mutants in ppGpp0 hosts that slow growth also lower ori/ter ratios. Our evidence can be taken to support the notion that ppGpp is the source of coordinate regulation of total cellular macromolecular content (protein, RNA and DNA) as a function of balanced growth rates. The mechanisms at play are now our focus since they remain largely unknown, especially for replication initiation.